The integrated processing of signals transduced by activating and inhibitory cell surface receptors regulates NK cell effector functions. Here, I review the structure, function, and ligand specificity of the receptors responsible for NK cell recognition.

Nk cell recognition

Munc13-4 Is Essential for Cytolytic Granules Fusion and Is Mutated in a Form of Familial Hemophagocytic Lymphohistiocytosis (FHL3)

Cell-surface receptors acquire information from the extracellular environment and coordinate intracellular responses Many receptors do not operate as individual entities, but rather as part of dimeric or oligomeric complexes Coupling the functions of multiple receptors may endow signaling pathways with the sensitivity and malleability required to govern cellular responses Moreover, multireceptor signaling complexes may provide a means of spatially segregating otherwise degenerate signaling cascades Understanding the mechanisms, extent, and consequences of receptor co-localization and interreceptor communication is critical; chemical synthesis can provide compounds to address the role of receptor assembly in signal transduction Multivalent ligands can be generated that possess a variety of sizes, shapes, valencies, orientations, and densities of binding elements This Review focuses on the use of synthetic multivalent ligands to characterize receptor function

Synthetic Multivalent Ligands as Probes of Signal Transduction

Granule exocytosis is the main pathway for the immune elimination of virus-infected cells and tumour cells by cytotoxic T lymphocytes and natural killer cells. After target-cell recognition, release of the cytotoxic granule contents into the immunological synapse formed between the killer cell and its target induces apoptosis. The granules contain two membrane-perturbing proteins, perforin and granulysin, and a family of serine proteases known as granzymes, complexed with the proteoglycan serglycin. In this review, I discuss recent insights into the mechanisms of granule-mediated cytotoxicity, focusing on how granzymes A, B and C and granulysin activate cell death through caspase-independent pathways.

The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal

The natural killer (NK)-cell immunological synapse is the dynamic interface formed between an NK cell and its target cell Formation of the NK-cell immunological synapse involves several distinct stages, from the initiation of contact with a target cell to the directed delivery of lytic-granule contents for target-cell lysis Progression through the individual stages is regulated, and this tight regulation underlies the precision with which NK cells select and kill susceptible target cells (including virally infected cells and cancerous cells) that they encounter during their routine surveillance of the body

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Formation and function of the lytic NK-cell immunological synapse

The cytolytic activity of NK cells is tightly regulated by inhibitory receptors specific for MHC class I Ags. We have investigated the composition of signal transduction molecules in the supramolecular activation clusters in the MHC class I-regulated cytolytic and noncytolytic NK cell immune synapses. KIR2DL3-positive NK clones that are specifically inhibited in their cytotoxicity by HLA-Cw*0304 and polyclonal human NK cells were used for conjugate formation with target cells that are either protected or are susceptible to NK cell-mediated cytotoxicity. Polarization of talin, microtubule-organizing center, and lysosomes occurred only during cytolytic interactions. The NK immune synapses were analyzed by three-dimensional immunofluorescence microscopy, which showed two distinctly different synaptic organizations in NK cells during cytolytic and noncytolytic interactions. The center of a cytolytic synapse with MHC class I-deficient target is comprised of a complex of signaling molecules including Src homology (SH)2-containing protein tyrosine phosphatase-1 (SHP-1). Closely related molecules with overlapping functions, such as the Syk kinases, SYK, and ZAP-70, and adaptor molecules, SH2 domain-containing leukocyte protein of 76 kDa and B cell linker protein, are expressed in activated NK cells and are all recruited to the center of the cytolytic synapse. In contrast, the noncytolytic synapse contains SHP-1, but is lacking other components of the central supramolecular activation cluster. These findings indicate a functional role for SHP-1 in both the cytolytic and noncytolytic interactions. We also demonstrate, in three-cell conjugates, that a single NK cell forms a cytolytic synapse with a susceptible target cell in the presence of both susceptible and nonsusceptible target cells.

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Spatial organization of signal transduction molecules in the NK cell immune synapses during MHC class I-regulated noncytolytic and cytolytic interactions.

Recent applications of imaging approaches and other methods of cell biology have provided high-resolution visualization of the location of fluorescent proteins in living and fixed cells during cell-cell interactions between lymphocytes, antigen presenting cells and target cells. We review the composition and dynamics of molecular and cytoskeletal events occurring during natural killer cell interactions with susceptible and nonsusceptible target cells. The natural killer cell immune synapse and the concomitant changes in cytoskeletal components and cytoplasmic organelles are described. The findings are compared with the observations made in T helper cells and cytotoxic T cells. It is concluded that the cytolytic immune synapses display spatial-temporal dynamics that are accelerated as compared with T helper cells. In addition, the cytolytic conjugates have unique characteristics relating to their effector function. Furthermore, the natural killer cell immune synapses in cytolytic and noncytolytic interactions are distinctly different and display patterns consistent with characteristic signaling pathways identified in biochemical studies of disrupted cells. The precise relationship between different stages of the natural killer cell immune synapse formation and progression in signal transduction pathways is yet to be established.

Visualization of signaling pathways and cortical cytoskeleton in cytolytic and noncytolytic natural killer cell immune synapses.

Inhibitory NK receptors with ligand specificity for MHC class I recruit Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) phosphatase and prevent autocytotoxicity Activation of SHP-1 depends upon Src kinase-mediated tyrosine phosphorylation of the cytoplasmic domain of the inhibitory receptor In this study we demonstrate, by quantitative temporal analysis, that talin, Lck, and SHP-1 are recruited to the synapse within 1 min in both cytolytic and noncytolytic conjugates Polarization of talin and Lck rapidly disappears in the noncytolytic interactions but persists in cytolytic interactions, where protein kinase C-θ, Src homology 2 domain-containing leukocyte protein of 76 kDa, and lysosomes are recruited within 5 min At 1 min SHP-1 clusters in the periphery of the cytolytic synapse, whereas it clusters in the center of the noncytolytic synapse Lck has multifocal distribution in both synapses consistent with the shared requirement for early tyrosine phosphorylation Our studies indicate that the spatial location of SHP-1 in the synapse distinguishes noncytolytic from cytolytic interactions within the first minute

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Cutting edge: differential segregation of the SRC homology 2-containing protein tyrosine phosphatase-1 within the early NK cell immune synapse distinguishes noncytolytic from cytolytic interactions.

Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.

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Ligand Binding to Inhibitory Killer Cell Ig-Like Receptors Induce Colocalization with Src Homology Domain 2-Containing Protein Tyrosine Phosphatase 1 and Interruption of Ongoing Activation Signals

Hina Maniar

Papers

Spatial organization of signal transduction molecules in the NK cell immune synapses during MHC class I-regulated noncytolytic and cytolytic interactions.

Visualization of signaling pathways and cortical cytoskeleton in cytolytic and noncytolytic natural killer cell immune synapses.

Cutting edge: differential segregation of the SRC homology 2-containing protein tyrosine phosphatase-1 within the early NK cell immune synapse distinguishes noncytolytic from cytolytic interactions.

Ligand Binding to Inhibitory Killer Cell Ig-Like Receptors Induce Colocalization with Src Homology Domain 2-Containing Protein Tyrosine Phosphatase 1 and Interruption of Ongoing Activation Signals